cardiovascular disease, single nucleotide polymorphisms; and the renin angiotensin system: is there...

SAGE-Hindawi Access to ResearchInternational Journal of HypertensionVolume 2010, Article ID 281692, 13 pagesdoi:10.4061/2010/281692

Review Article

Cardiovascular Disease, Single Nucleotide Polymorphisms; andthe Renin Angiotensin System: Is There a MicroRNA Connection?

Terry S. Elton,1, 2, 3 Sarah E. Sansom,1 and Mickey M. Martin1

1 Davis Heart and Lung Research Institute, The Ohio State University, DHLRI 515, Columbus, OH 43210, USA2 Division of Pharmacology, College of Pharmacy, The Ohio State University, OH 43210, USA3 Department of Medicine, College of Medicine, Division of Cardiology, The Ohio State University, OH 43210, USA

Correspondence should be addressed to Terry S. Elton, [email protected]

Received 4 May 2010; Accepted 25 June 2010

Academic Editor: Stephen B. Harrap

Copyright © 2010 Terry S. Elton et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Essential hypertension is a complex disorder, caused by the interplay between many genetic variants, gene-gene interactions, andenvironmental factors. Given that the renin-angiotensin system (RAS) plays an important role in blood pressure (BP) control,cardiovascular regulation, and cardiovascular remodeling, special attention has been devoted to the investigation of single-nucleotide polymorphisms (SNP) harbored in RAS genes that may be associated with hypertension and cardiovascular disease.MicroRNAs (miRNAs) are a family of small, ∼21-nucleotide long, and nonprotein-coding RNAs that recognize target mRNAsthrough partial complementary elements in the 3′-untranslated region (3′-UTR) of mRNAs and inhibit gene expression bytargeting mRNAs for translational repression or destabilization. Since miRNA SNPs (miRSNPs) can create, destroy, or modifymiRNA binding sites, this review focuses on the hypothesis that transcribed target SNPs harbored in RAS mRNAs, that altermiRNA gene regulation and consequently protein expression, may contribute to cardiovascular disease susceptibility.

1. Introduction

Identifying the genes and mutations that contribute todisease is a central aim in human genetics. Single nucleotidepolymorphisms (SNPs) are mutations that occur at genomepositions at which there are two distinct nucleotide residues(alleles) that each appear in a significant portion (i.e., aminor allele frequency greater than 1%) of the humanpopulation [1]. There are some estimated 14 million SNPs[2] in the human genome that occur at a frequency ofapproximately one in 1,200–1,500 bp [3]. SNPs can affectprotein function by changing the amino acid sequences(nonsynonymous SNP) or by perturbing their regulation(e.g., affecting promoter activity [4], splicing process [5],and DNA and pre-mRNA conformation). When SNPs occurin 3′-UTRs, they may interfere with mRNA stability andtranslation by altering polyadenylation and protein/mRNAregulatory interactions. Recently, a new layer of posttran-scriptional miRNA-mediated gene regulation has been dis-covered and shown to control the expression levels of a largeproportion of genes (reviewed in [6]). Importantly, SNPs in

microRNA (miRNA) target sites (miRSNPs) represent a spe-cific class of regulatory polymorphisms in the 3′-UTR thatmay lead to the dysregulation of posttranscriptional geneexpression. Thus, for miRNAs acting by this mechanism, themiRSNPs may lead to heritable variations in gene expression.Given that the renin angiotensin system (RAS) is intricatelyinvolved in the pathogenesis of cardiovascular disease [7–12],we review and discuss the presently available evidence formiRSNPs-mediated RAS gene regulation and its importancefor phenotypic variation and disease.

2. Current View ofthe Renin Angiotensin System

The RAS plays a critical role in regulating the physiologicalprocesses of the cardiovascular system [reviewed in [7–14]].The primary effector molecule of this system, angiotensinII (Ang II), has emerged as a critical hormone that affectsthe function of virtually all organs, including heart, kidney,vasculature, and brain, and it has both beneficial and

2 International Journal of Hypertension

ACEACE

ACE2

ACE2

Renin

Am A

Am B/N

C C

CCCC

N

NN

NN

N

Angiotensinogen

Angiotensin I

Angiotensin II

Angiotensin III Angiotensin IV

PRR MasR

VasoconstrictionHypertrophyProliferation

Pro-oxidationProfibrosis

VasodilationAntihypertrophyAntiproliferation

AntioxidationAntifibrosis

HypertensionCardiac/vascular remodeling

Atherosclerosis

AntihypertensiveAntiremodeling

Antiatherosclerosis

AT1R AT2R AT4R/IRAP

Angiotensin (1–9)

Angiotensin (1–7)

Figure 1: Summary of the RAS incorporating the Ang peptide family and physiological effects mediated via ATR subtypes. Under theclassical RAS schema, Ang II is produced, via renin and ACE, to act with equal affinity on two ATR subtypes, AT1R and AT2R (large arrows).However, it is now appreciated that a number of breakdown products of Ang II, namely, Ang (1–7), Ang III, and Ang IV exert their ownunique effects that are distinct (and often opposite) to those of Ang II. Such effects are often mediated via newly recognized receptors suchas MasR for Ang (1–7) and AT4R (also known as IRAP) for Ang IV, or additionally via AT2R stimulation. ACE2 is also a new pathway forthe formation of Ang (1–7). Newly identified Ang receptor binding proteins associated with different ATR subtypes may also modify ATRactivation. Thus, overstimulation of AT1R (and PRR) by Ang II, which can contribute to a plethora of cardiovascular disease processes, maybe counter-regulated by a number of non-AT1R mechanisms. Most notably, AT2R stimulation usually causes opposing effects to AT1R, asindicated. It is also likely that the MasR exerts a similar counter-regulatory role whereas the evidence is more preliminary and speculativefor AT4R/IRAP. In terms of mediators, Ang II itself stimulates AT2R whereas the shorter Ang peptides stimulate their cognate receptors andpossibly also AT2R.

pathological effects [7–14]. Acute stimulation with Ang IIregulates salt/water homeostasis and vasoconstriction, mod-ulating blood pressure, while chronic stimulation promoteshyperplasia and hypertrophy of vascular smooth musclecells (VSMCs). In addition, long-term exposure to Ang IIalso plays a pathophysiological role in cardiac hypertro-phy and remodeling, myocardial infarction, hypertension,atherosclerosis, in-stent restenosis, reduced fibrinolysis, andrenal fibrosis [7–14].

Ang II, an octapeptide hormone, is produced sys-temically via the classical RAS and locally via the tissueRAS [7–14]. In the classical RAS, circulating renal-derivedrenin cleaves hepatic-derived angiotensinogen to form thedecapeptide angiotensin I (Ang I), which is converted byangiotensin-converting enzyme (ACE) in the lungs to thebiologically active Ang II (Figure 1). Alternatively, a recentlyidentified carboxypeptidase, ACE2, cleaves one amino acid

from either Ang I or Ang II [15–18], decreasing Ang II levelsand increasing the metabolite Ang 1–7, which has vasodilatorproperties. Thus, the balance between ACE and ACE2 isan important factor controlling Ang II levels [15–18]. AngII is also further degraded by aminopeptidases to Ang III(Ang 2–8) and Ang IV (Ang 3–8) (Figure 1) [7]. Althoughthe RAS was originally regarded as a circulating system,many of its components are localized in tissues, includingthe heart, brain, blood vessels, adrenal, kidney, liver andreproductive organs, indicating the existence of local tissueRASs [19]. In addition to ACE-dependent pathways of AngII formation, non-ACE pathways have also been described.Chymotrypsin-like serine protease (chymase) may representan important mechanism for conversion of Ang I to AngII in the human heart, kidney, and vasculature and maybe particularly important in pathological conditions such ascoronary heart disease [20].

International Journal of Hypertension 3

The biological responses to Ang II are mediated byits interaction with two distinct high-affinity G protein-coupled receptors (GPCRs) designated AT1R and AT2R(Figure 1) [7]. Both AT1R and AT2R possess similar affinityfor Ang II [21]; however, pharmacologically, these receptorscan be distinguished according to inhibition by specificantagonists. For example, AT1R are selectively antagonizedby biphenylimidazoles such as losartan (angiotensin receptorblockers, ARB) [21] whereas tetrahydroimidazopyridinessuch as PD123319 specifically inhibit AT2R [21, 22]. Inter-estingly, all of the classical actions of Ang II, includingvasoconstriction, effects on fluid and electrolyte homeostasis,and influences on cellular growth and differentiation, havebeen shown to be due to stimulation of AT1R located on theplasma membrane of cells [7–12]. Additionally, the majorityof the pathophysiological effects (i.e., cardiac hypertrophyand remodeling, myocardial infarction, hypertension, etc.)of Ang II are also mediated via the AT1R [7–12]. In contrast,it is thought that the AT2R counter-regulates AT1R function(reviewed in [13, 14]). It is also speculated that duringcardiovascular disease, AT2R upregulation and activation byAng II, or angiotensin peptide fragments (i.e., Ang III, AngIV, and/or Ang 1–7) may limit AT1R-mediated overactivityand cardiovascular pathologies [13, 14].

Although the AT1R and AT2R have been intensivelyinvestigated it is now clear that angiotensin fragments canbind to and activate other receptor subtypes. For example,Ang 1–7 acts on the Mas GPCR (MasR) and has vasodilatoryand antiproliferative effects. This arm of the RAS is alsothought to counterbalance the effects of Ang II acting onthe AT1R (Figure 1) (reviewed in [18]). Additionally, AngIV can bind to the angiotensin II type 4 receptor (AT4R)or the membrane-bound, insulin-regulated aminopeptidase(IRAP) (Figure 1) and mediate the enhancement of cognitivefunction, modulate blood flow, increase natriuresis, inhibitcardiomyocyte hypertrophy, and improve endothelial func-tion in animal models of atherosclerosis [23, 24].

Finally, recent studies now suggest that renin, the aspartylprotease that cleaves angiotensinogen into Ang I, andprorenin, its proenzyme inactive form, can bind to what isnow designated as the (pro)renin receptor (PRR) (reviewedin [25–27]). Interestingly, the binding of renin/prorenin toPRR has been shown to have two major consequences. First,the binding of renin to its receptor increases angiotensinogenconversion to Ang I by five-fold, and prorenin, which isvirtually inactive in solution, also displays enzymatic activityfollowing receptor binding [25–27]. Second, receptor-boundrenin/prorenin activates the MAP kinases ERK1/2 andp38 pathways, which in turn, leads to the upregulationof profibrotic and cyclooxygenase-2 genes independent ofAng II generation [25–27]. Therefore, the activation andpotentiation of renin/prorenin enzymatic activity, togetherwith specific PRR-mediated signaling, could have strikingeffects on cardiovascular regulation. Taken together, thesestudies suggest that RAS is unexpectedly complex andmultilayered. New components and functions of the RASare still being unraveled and the physiological significance,and ultimately the clinical relevance, of these factors remainlargely undefined.

3. Overview of miRNA Biology

MicroRNAs (miRNAs) are endogenous, short (20–23nucleotide), and single-stranded nonprotein-coding RNAmolecules that regulate gene expression (reviewed in [28]).These molecules act by binding to their target mRNAs,preferentially to the 3′-UTR, using a partial base-pairingmechanism. In order for a miRNA to give rise to functionalconsequences, the 7-8 nucleotides (nt) at the most 5′ endmust have exact complementarity to the target mRNA,generally referred to as the “seed” region [29]. The currentmodel for inhibition of expression by a miRNA suggeststhat a miRNA either inhibits translation or induces degra-dation of its target mRNA, depending upon the overalldegree of complementarity of the binding site, number ofbinding sites, and the accessibility of those binding sites[30–32].

In mammals, computational predictions indicate thatmiRNAs may regulate 60% of all human protein codinggenes [33], and have been increasingly implicated in thecontrol of various biological processes, including cell dif-ferentiation, cell proliferation, development and apoptosis,and many pathological processes such as cancer, Alzheimer’sdisease, and cardiovascular disease [34–36]. There are esti-mated to be >1,000 miRNAs encoded by the human genome[37, 38], each of which can act on multiple target mRNAs.Conversely, individual mRNAs are commonly targeted bymultiple miRNAs, which results in a combinatorial repres-sion of gene expression more robust than the suppressionthat results from a single miRNA [39, 40]. Although miRNAsare known to mediate posttranscriptional gene silencing inthe cytoplasm, recent evidence indicates that at least somefraction of mammalian miRNAs may also activate or inhibitgene expression at the transcriptional level [41, 42]. Takentogether, these miRNA phenomena allow for enormouscombinatorial complexity and regulatory potential.

4. miRNA Biogenesis

Mature miRNAs are processed from primary miRNA tran-scripts (pri-miRNAs), which are either transcribed fromindependent miRNA genes or are portions of introns ofprotein-coding RNA polymerase II transcripts (Figure 2)[43–45]. miRNAs tend to cluster throughout the genome andmany of these clusters are likely transcribed as polycistrons[46–48]. Although little is known regarding the regulationof miRNA transcription, it is recognized that miRNAexpression is usually regulated by established transcriptionalmechanisms. Interestingly, however, it has been shown thateach miRNA located within the same genomic cluster maybe transcribed and regulated independently [49].

During the transcriptional process, pri-miRNAs foldinto hairpin structures containing imperfectly base-pairedstems and are endonucleolytically cleaved by the nuclearmicroprocessor complex formed by the RNase III typeendonuclease Drosha and the DiGeorge critical region8 (DGCR8) protein [50]. The Drosha/DGCR8 complexprocesses pri-miRNAs into ∼70-nucleotide hairpins knownas pre-miRNAs (Figure 2) [28, 51]. In animals, pre-miRNAs


TRBP

Drosha

DGCR8

RNA Pol II/III

Dicer

AGO2

AAAAA

AAAAA

3´

3´

3´

3´

3´

3´

3´

3´

3´

5´

5´

5´

5´

5´

5´

5´

5´

5´

CytoplasmNucleus

MicroRNA gene or intron

Transcription

Pri-miRNA

Processing

Pre-miRNA

AGO1–4

AGO1–4

Exportin-5

Maturation

Strand selection andRISC formation

mRNA target cleavage

Translational repressionand/or deadenylation

Decreased mRNA targetprotein levels

Figure 2: Schematic representation outlining miRNA biogenesis including transcription, maturation, and miRNA/mRNA targeting. Thisdiagram also outlines two potential mechanisms for miRNA/mRNA silencing. The specific details discussing these processes are included inthe text.

are exported from the nucleus to the cytoplasm via Exportin-5, where they are cleaved by Dicer complexed with theTAR RNA binding protein (TRBP), to yield ∼20-bp miRNAduplexes. In principle, the miRNA duplex could give rise totwo different mature miRNAs. However, in a manner similarto siRNA duplexes, only one strand is usually incorporatedinto miRNA-induced silencing complexes (miRISCs) andguides the complex to target mRNAs; the other strandis degraded (the complementary miRNA∗ strand) [52].This functional asymmetry depends on the thermodynamicstability of the base pairs at the two ends of the duplex, withthe miRNA strand, which has the least stable base pair at its5′ end in the duplex, being loaded into the miRISC [53]. Still,recent data suggest that both arms of the pre-miRNA hairpincan give rise to mature miRNAs [28, 45, 51].

5. miRNA/mRNA Silencing

Although the details are not well understood, pre-miRNAprocessing by Dicer is coupled with the assembly ofmiRNAs into ribonucleoprotein complexes called micro-RNPs (miRNPs) or miRISCs [28, 51, 54]. One of the keycomponents of miRNPs is the Argonaute (AGO) proteinfamily, AGO1 to AGO4, and while all four AGO proteinsfunction in miRNA repression only AGO2 functions in

mRNA target cleavage (Figure 2) [54, 55]. Once the miRNAis processed, the mature miRNA acts as an adaptor formiRISC to specifically recognize and regulate particularmRNAs. It is currently thought that the miRNA-loaded RISCis targeted to a given mRNA by a mechanism where themiRISC binds many sites nonspecifically until the correcttarget site is found [56].

With few exceptions, miRNA binding sites in animalmRNAs are present in the 3′-UTR and mature miRNAsbase pair with their target mRNAs imperfectly, following aset of rules that have been identified by experimental andbioinformatics analyses [29, 57–60]. First, miRNA/mRNAtarget recognition involves Watson-Crick base pairing thatmust be perfect and contiguous at the 5′-end of the miRNAfrom nucleotides 2 to 8. This section represents the “seed”region and nucleates the miRNA-mRNA association. Second,G:U wobble pairing in the seed sequence is highly detrimen-tal to miRNA function despite its favorable contribution toRNA:RNA duplexes. Third, an A residue across position 1of the miRNA, and an A or U across position 9, improvethe site efficiency, although they do not need to base pairwith miRNA nucleotides. Fourth, it has been established thatmiRNAs that have suboptimal 5′ Watson-Crick base pairingneed substantial complementarity to the miRNA 3′ half tostabilize the interaction and to be functional. Finally, thecontext of the miRNA’s binding sites harbored in the 3′-UTR


of target mRNAs, also influence the functional importanceof these sites [61]. For example, miRNA site efficacy can beimproved if the site is positioned at least 15 nt downstreamfrom the stop codon, away from the center of long 3′-UTRs,and near AU-rich nucleotide regions. These factors can makethe 3′-UTR regions less structured and hence more accessibleto miRNP recognition.

When an endogenous miRISC programmed with miRNAbinds to a recognition site that is perfectly complementary,this target mRNA will be cleaved by the miRISC (Figure 2)[56, 62–65]. In contrast, miRISCs that are imperfectlymatched with target mRNAs can repress translation initia-tion at either the cap-recognition stage [66–70] or the 60Ssubunit joining stage [71]. Alternatively, binding of miRISCscan induce deadenylation and decay of target mRNAs [62].

6. Computational Algorithms toPredict miRNA/mRNA Targets

Computational miRNA/mRNA target programs remain theonly source for rapid prediction of miRNA recognition sitesharbored within the 3′-UTR of target mRNAs. Therefore,the development of reliable computational target predictionprograms is critical in advancing our understanding ofmiRNA function. Given that miRNA functionality usuallyrequires seed sequence complementarity [28, 61] the mainprediction feature used in most of these programs is thesequence alignment of the miRNA seed to the 3′-UTR of can-didate target genes. Additionally, many current algorithmsalso utilize conservation of miRNA/mRNA target sites acrossspecies as an important parameter for the identification ofbona fide targets; notably however, the conservation of amiRNA binding site harbored in a given mRNA target is nota requirement for a functional miRNA.

A recent review article [72] comparing eight of themost commonly used algorithms for miRNA target pre-diction for the human and mouse genome programsdemonstrated that the four top algorithms, DIANA-microT 3.0 (http://microrna.gr/microT) [73], TargetScan 5.0(http://www.targetscan.org/) [74], Pictar (http://pictar.org/)[75], and ElMMo (from http://www.mirz.unibas.ch) [76] allhave a precision of ∼50% with a sensitivity that ranges from6% to 12%. Of the top four performing programs, it isimportant to note that TargetScan is the most up-to-dateregarding the number of miRNAs and genes used and Pictaris least updated [72]. Most investigators assume that mRNAtargets predicted by more than one algorithm are moreaccurate than other targets thus leading to higher predictionprecision. However, Alexiou et al. [72] demonstrated thatmany of the algorithm combinations performed worse thanthe prediction of a single algorithm. These investigatorsreason that the better specificity of a combination is achievedby a higher price for the sensitivity. Taking this intoaccount, our laboratory has analyzed all of the classical andnonclassical RAS components for putative miRNA bindingsites by the TargetScan algorithm (Table 1). Importantly, thisanalysis suggests that miRNAs may play a major role inregulating the expression of RAS proteins.

Although many programs are available online for theprediction of individual mRNA targets of miRNAs (seeabove), the identification of authentic mRNA targets remainsproblematic. Mammalian miRNAs bind to the mRNA withimperfect complementarity thus how binding sites arerecognized is only partially understood. Therefore, somebioinformatically predicted targets turn out to be falseand others are entirely overlooked. Experimental validationof targets is therefore an important step in defining thefunctions of individual miRNAs (for review, see [77]).

7. Experimentally ValidatedmiRNA/RAS Targets

Although classical and nonclassical RAS components harborputative miRNA binding sites, very few of these siteshave been experimentally validated. Our laboratory hasdemonstrated that miR-155 specifically interacted withthe algorithm-predicted binding site harbored in the 3′-UTR of the human AT1R (hAT1R) mRNA (Table 1) [78,79]. Additionally, miR-155 gain-of-function experiments(i.e., cells were transfected with partially double-strandedRNAs that mimic the Dicer cleavage product and aresubsequently processed into their respective mature miR-NAs) inhibited the expression of the hAT1R and alsoattenuated Ang II-induced signaling via the hAT1R infibroblasts and vascular smooth muscle cells (VSMCs)[78, 79]. These results also demonstrated that transfectionwith miR-155 did not significantly decrease hAT1R steadystate mRNA levels, suggesting that miR-155 can decreasehAT1R expression by inhibiting translation of the mRNA,rather than targeting it for degradation. In contrast, loss-of-function experiments (i.e., cells were transfected withmiRNA inhibitors; antisense single-stranded chemically-enhanced oligonucleotides, ASO) demonstrated that trans-fection of anti-miR-155 not only increased hAT1R expres-sion but also enhanced Ang II-induced signaling via thehAT1R, indicating that miR-155 plays a physiological rolein regulating the expression of hAT1Rs in human fibroblastsand VSMCs [78, 79]. Recently, our laboratory also demon-strated that hAT1R expression can be regulated by miR-802[80].

In support of the miR-155/hAT1R studies describedabove, trisomy 21 (Ts21) mediated overexpression of miR-155 (the bic/miR-155 gene is located on human chromosome21 and is triplicated in Down syndrome [DS] individuals)[81, 82] resulted in the attenuation of hAT1R protein levelsin fibroblasts isolated from one monozygotic twin with DSwhen compared to fibroblasts isolated from the unaffectedeuploid twin [83]. Interestingly, individuals with DS havesignificantly lower systolic and diastolic blood pressures[84–87], a reduced risk of vascular anomalies [88], anda low prevalence of coronary artery disease [89–93] whencompared with the general population. Given that the over-expression of miR-155 in Ts21 results in attenuated hAT1Rprotein levels, we speculate that this may be one mechanismwhich contributes to the lack of cardiovascular diseaseobserved in individuals with DS [84–87].


Table 1: TargetScan∗ algorithm-predicted RAS putative miRNA/mRNA target sites.

RAS Component Total Conserved miRNA Targets Total Poorly Conserved miRNA Targets Overall Total miRNA Targets

AGTR1 (AT1R) 0 56 56

AGTR2 (AT2R) 2 96 98

ACE 0 26 26

ACE2 1 57 58

AGT 1 31 32

REN 0 15 15

ATP6AP2 (PRR) 4 49 53

MAS1 (MasR) 0 12 12

LNPEP (AT4R) 5 42 47∗

Source: TargetScan (April, 2009). The number of potential targets is dramatically dependent upon the algorithm utilized.

Finally, Boettger et al. [94] demonstrated by genomic,proteomic, and transcriptional analyses that mouse ACEmRNA is a miR-145 target. The miR-143/-145 gene clusterincludes miR-143 and miR-145, which lie within a 1.7-kbhighly conserved region of mouse chromosome 18 [94].miRNA microarray hybridization experiments revealed thatmiR-143 and miR-145 are enriched in murine vascularsmooth muscle cells (VSMCs) [94, 95]. In the mouseembryo, miR-143/-145 expression is restricted to heart, vas-cular, and visceral SMCs [94, 95]. During late fetal and post-natal development, miR-143/-145 expression is downregu-lated in the heart but persists in vascular and visceral SMCs.Homozygous miR-143/-145 knockout mice were viable butexhibited thinning of the arterial tunica media (muscularlayer), with a reduction in the number of contractile VSMCsand a concomitant increase in the number of proliferativeVSMCs [94]. Physiological characterization of miR-143/-145 deficient mice and arterial segments from these animalsrevealed defects in Ang II-induced VSMC contractility andhomeostatic control of blood pressure. Consistent with thisobservation, pharmacological inhibition of ACE or the AT1Rpartially reversed vascular dysfunction and normalized geneexpression in the mutant mice [94]. Since miR-145 regulatesthe expression of ACE, when the miR-143/-145 gene clusteris mutated, the levels of membrane-bound ACE in VSMCsincrease, causing the chronic stimulation of VSMCs by AngII, which in turn results in desensitization and “angiotensinresistance” of VSMCs. Given that miR-143/145 mutant micedeveloped neointimal lesions in the absence of hyperlidemia,lipid depositions, and foam cells also highlights the potentialrole of VSMCs in the pathogenetic process leading toatherosclerosis. The enhancement of Ang II signaling due tothe increased expression of ACE would certainly contributeto this process, since increased levels of Ang II have beenshown to promote atherosclerotic lesions in apoE-deficientmice [96].

Although the hAT1R and mouse ACE are experimentallyvalidated targets of miRNAs, it is important to note thatnone of these recognition sites are conserved across species(Table 1). Therefore, although miR-155 and -802 represshAT1R expression in humans, these miRNAs will not lead tothe repression of AT1R levels in mice or rats. Likewise, miR-

145 will repress ACE expression in mice but will not regulateACE levels in humans.

8. miRSNPs

Since a large number of miRNA binding sites are harboredin the 3′-UTRs of RAS component mRNAs (Table 1), thereis a high probability that SNPs will occur within miRNAtarget sites. By definition, miRSNPs have the potential tocreate, destroy, or modify the efficiency of miRNA binding,if the SNP occurs in the seed region (i.e., the region ofbase-pairing between nucleotides 2 and 8 of the miRNA andcomplementary nucleotides in the target mRNA) [97–99].Based on this definition, there are two mechanisms by whichmiRSNPs can be functionally important: as a gain- or as aloss-of-function variation. A gain-of-function effect wouldresult if the SNP enhances the targeting of the miRNA orcreates a new target site in the 3′-UTR of the mRNA. Inthis scenario, protein expression of the target mRNA wouldbe attenuated. In contrast, a loss-of-function effect wouldresult when the SNP decreases or abolishes the interactionof the miRNA with its mRNA target, thus resulting inan augmentation of protein expression. In support of thisconclusion, miRSNPs have been implicated in Tourettesyndrome [100], papillary thyroid cancer [101], muscularityin sheep [102], hereditary spastic paraplegia type 31 [103],methotrexate resistance [104], breast cancer [105], andtumor susceptibility [106]. These examples include bothgain- and loss-of-function miRSNPs.

9. The Human AT1R Gene and miRSNPs

The hAT1R gene has been found to be highly polymorphic[107]. In particular, a SNP has been described in which thereis an A/C transversion at position +1166 (i.e., 1166 base-pairs downstream from the start codon, dsSNP# rs5186)located in the 3′-UTR of the hAT1R gene. The increasedfrequency of the +1166 C-allele has been associated withhypertension [108–115], cardiac hypertrophy [116–118],aortic stiffness [119–121], myocardial infarction [122], heartfailure [123–125], abdominal aortic aneurysms [126, 127],


3´

3´

G G G G A U A G U G C U A A U C G U A A U U

5´

5´

U U C A C U A C C A - A A U G A G C A U U A G . .. .

miR-155

hAT1R mRNA(A-allele)

(a)

3´

3´5´

5´

. .. .

G G G G A U A G U G C U A A U C G U A A U U

U U C A C U A C C A - A A U G A G C C U U A G

miR-155

hAT1R mRNA(C-allele)

(b)

Figure 3: The human AT1R +1166 A/C SNP occurs in the miR-155-binding site. (a) Complementarity between miR-155 and the hAT1R3′-UTR site targeted (70–90 bp downstream from the human AT1R stop codon). The +1166 A/C SNP corresponds to the nucleotide86 bp downstream from the human AT1R stop codon (shown in red print). The binding of miR-155 to the hAT1R 3′-UTR target sitefulfills the requirement of a 7-bp seed sequence of complementarity at the miRNA 5′ end when the +1166 A-allele is expressed. (b)Complementarity between miR-155 and the human AT1R 3′-UTR harboring the +1166 C-allele. If the +1166 C-allele is expressed, theseed sequence requirement would not be met and, as a consequence, it would be expected that human AT1R expression would be elevated[79].

and increased oxidative stress levels in human heart failure[128]. However, the physiological relevance of this poly-morphism is uncertain because of its location within thenoncoding region of the hAT1R gene. Since our laboratoryhas previously demonstrated that miR-155 interacted with aspecific cis-response element localized in the hAT1R 3′-UTR[78], we investigated whether or not there was a correlationbetween the +1166 A/C SNP and the miR-155-binding site[79, 83]. Importantly, computer alignment revealed that the+1166 A/C SNP occurs within the cis-response element atthe site where miR-155 was shown to interact (Figure 3).The interaction between miR-155 and the hAT1R 3′-UTRharboring the A-allele fulfills the seed sequence rules [29]since there is a 7-bp region of complementarity betweenthe 5′ end of miR-155 and the hAT1R mRNA target site(Figure 3(a)). In contrast, if a hAT1R mRNA that harborsthe +1166 C-allele is expressed, the complementary seedsite is interrupted (Figure 3(b)), and the thermodynamicsof the miRNA:mRNA duplex would be significantly altered(i.e., a decrease in free energy) [79]. Therefore, the presenceof the +1166 C-allele miRSNP would decrease the abilityof miR-155 to interact with the cis-regulatory site locatedin the hAT1R 3′-UTR. As a consequence, it would beexpected that aberrantly high levels of the hAT1R would besynthesized. In support of this hypothesis we demonstratedthat when the hAT1R cis-response element harboring the C-allele was present in luciferase mRNAs, the ability of miR-155 to inhibit luciferase activity was significantly attenuated[79]. When identical experiments were performed utilizingmutant miR-155, which restored perfect Watson-Crick com-plementarity, luciferase activity was decreased to levels thatwere comparable with experiments utilizing the hAT1R cis-response element harboring the A-allele and miR-155 [79].To further demonstrate that the presence of the +1166 C-allele can influence hAT1R density, expression constructs thatproduced hAT1R mRNAs containing either the A- or C-allelewere cotransfected with miR-155 or mut-miR-155. Theseexperiments again demonstrated that when seed sequence

complementarity was not fulfilled, regardless of whethermiR-155 or mutant miR-155 was utilized, hAT1R levelswere always higher than the levels obtained when perfectcomplementarity was present between the miRNA and thehAT1R cis-response element [79]. Taken together, thesestudies provide the first feasible biochemical mechanism bywhich the +1166 A/C polymorphism (i.e., miRSNP) canlead to increased hAT1R densities and possibly cardiovasculardisease.

10. miRSNPS and RAS Component Genes

To begin to investigate whether other RAS componentsharbored SNPs in their 3′-UTRs which created miRSNPs,the SNP Geneview Report (http://www.ncbi.nlm.nih.gov/nucleotide/) for each gene was surveyed and all of the iden-tified SNPs were analyzed utilizing the Patrocles algorithm(http://www.patrocles.org/) [99]. Specifically the “PatroclesFinder” was utilized since it allows one to compare twosequences and subsequently determines the miRNA bindingsites that are different between the two sequences. Thus,a region of the transcribed 3′-UTR which harbored thecommon allele was compared with the same region contain-ing the mutated allele (select motif length: human 7 mers)and the generation of putative miRSNPs was determined(Table 2). Importantly, this analysis demonstrated that the3′-UTRs of the RAS components harbor a number ofmiRSNPs some of which alter or destroy legitimate miRNAbinding sites and others that create novel, illegitimatetarget sites. Hypothetically, if a loss-of-function miRSNPoccurred in the AGTR1, ACE, AGT, REN, or ATP6AP2 gene,an increased incidence of hypertension, cardiac/vascularremodeling, and atherosclerosis would be observed (Table 3).In contrast, if a loss-of-function miRSNP occurred inthe AGTR2, ACE2, MAS1, or LNPEP gene, a decreasedincidence of hypertension, cardiac/vascular remodeling, andatherosclerosis would be expected (Table 3).


Table 2: Patrocles∗ algorithm-predicted RAS component miRSNPs.

RAS Component dbSNP# Hetero dbSNP bp from Stop Codon Loss of Function miRSNP Gain of Function miRSNP

AGTR1 (AT1R)

rs5184 0.069 A>G 2 miR-668

rs56343250 N.D. 40 mR-1237, -1248

rs5185 0.130 T>G 70 miR-302c, -573 miR-143∗, -301a

rs12721277 0.028 G>A 82 miR-579

rs5186 0.500 A>C 86 miR-155

rs5187 N.D. A>G 93 miR-562, -548n miR-646

rs1799870 0.055 C>T 135

rs5188 0.109 G>A 317 miR-1197

rs55707609 N.D. A>T 335 miR-299-3p

rs5189 0.500 G>T 437 miR-570

rs12721276 0.061 C>A 461 miR-128, -27a

rs12721275 0.055 C>T 484 miR-143∗

rs12721274 0.028 T>C 556 miR-641

rs440881 N.D. A>C 565 miR-30b

rs1051649 N.D. C>T 680 miR-1197 miR-7

rs35533650 N.D. A>G 704

rs380400 0.500 A>G 798

rs35393661 N.D. ->AT 803

AGTR2 (AT2R)

rs34589510 N.D. ->T 57

rs5193 0.241 G>T 199

rs5194 0.499 A>G 205 miR-1229

rs11091046 0.496 A>C 501

rs17231436 0.131 G>C 581 miR-384

rs41312570 N.D. C>T 645 miR-301a, -130a

rs17231443 0.025 C>G 816 miR-548f, -570

rs17237806 0.050 C>T 837

rs12858432 N.D. C>T 906

rs12845035 0.030 C>G 1103 miR-361-3p

rs17237820 0.073 A>T 1111 miR-548a-3p

rs17231478 0.038 G>T 1185

rs17237827 0.025 A>G 1274

rs17231450 0.050 C>A 1318 miR-150∗

rs17231457 0.050 A>C 1536 miR-571

ACE None

ACE2 None

AGT

rs61762526 0.007 G>T 33

rs5042 0.008 C>T 40 miR-486-3p

rs4753 0.021 G>C 158 miR-337-5p

rs61751079 0.002 G>A 159 miR-639, -539

rs5043 0.021 T>C 167

rs61751080 0.002 T>A 168

rs11684 N.D. C>T 184 miR-103, -107

rs1803103 N.D. G>T 187 miR-483-3p

rs15022 N.D. G>T 188 miR-483-3p

rs1803104 N.D. A>G 316

rs1803106 N.D. A>C 338 miR-129-5p miR-335∗

rs61751081 0.002 C>T 350

rs5044 0.029 T>G 423 miR-1283, -606

rs61762525 0.004 C>T 473 miR-548l, -559

rs7079 0.312 C>A 556 miR-218-1∗, -584

rs55720804 0.035 C>- 573


Table 2: Continued.

RAS Component dbSNP# Hetero dbSNP bp from Stop Codon Loss of Function miRSNP Gain of Function miRSNP

rs61751082 0.023 C>- 575

REN rs11799601 0.500 C>A 48 miR-326, -330-5p

r11571124 0.041 C>T 145 miR-138 miR-150∗

ATP6AP2 (PRR) rs5963816 0.296 A>T 266 miR-664

rs6609080 0.186 A>G 358 miR-1179

rs9062 N.D. G>T 654 miR-508-5p miR-410

rs10536 0.334 A>G 761 miR-802 miR-140-3p, -497∗

rs1060063 0.340 T>C 809

MAS1 (MasR) None

LNPEP (AT4R)

rs17087239 0.004 C>T 61 miR-992, -15b

rs39602 0.488 C>G 217

rs75912980 0.180 T>A 364 miR-1225-5p, -9

rs1057808 0.014 T>C 408 miR-922, -15b

rs3756618 0.028 A>T 501 miR-22∗, -26b∗ miR-223

rs35838718 N.D. A>- 561

rs79818663 0.105 G>T 594

rs77639920 N.D. C>T 614 miR-664∗

rs62377081 N.D. G>T 695 miR-302a∗, -1264 miR-548l∗

Source: http://www.patrocles.org/.

Table 3: Physiological ramifications of RAS miRSNPs.

RAS Component Loss of FunctionmiRSNP

Physiological ResultGain of

FunctionmiRSNP

Physiological Result

AGTR1 (AT1R) AT1R↑HypertensionCardiac/vascularremodelingAtherosclerosis

AT1R↓AntihypertensiveAntiremodelingAnti-atherosclerosis

ACE ACE↑ ACE↓AGT AGT↑ AGT↓REN REN↑ REN↓ATP6AP2 (PRR) PRR↑ PRR↓AGTR2 (AT2R) AT2R↑

AntihypertensiveAntiremodelingAntiatherosclerosis

AT2R↓ HypertensionCardiac/vascularremodelingAtherosclerosis

ACE2 ACE2↑ ACE2↓MAS1 (MasR) MasR↑ MasR↓LNPEP (AT4R) AT4R↑ AT4R↓

11. Conclusion

miRNAs have been increasingly implicated in the control ofvarious biological processes, including cell differentiation,cell proliferation, development and apoptosis, and manypathological processes such as cancer, Alzheimer’s disease,and cardiovascular disease [34–36]. Importantly, severalstudies which have now demonstrated that polymorphismsat the miRNA target site in the 3′-UTRs of transcribedmRNAs can have detrimental effects given that miRSNPscan lead to the modulation of gene expression [79, 83, 100–106]. In support of this hypothesis, a recent study suggestedthat differences in SNP allele frequency among ethnic groupsaccount for differences in gene expression [129]. Therefore,we speculate that miRSNPs can modulate RAS phenotypicgene expression diversities, at least in part, through alterationof miRNA target binding capability, ultimately leading to

differences in the susceptibility to complex genetic disorders,such as cardiovascular disease.

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